Analytical method development and validation for simultaneous estimation of Fimasartan Potassium Trihydrate and Cilnidipine in synthetic mixture by HPLC for the treatment of hypertension stage-II

A specific, accurate, precise, robust, and cost-effective HPLC method was developed and validated for quantitative analysis of Fimasartan Potassium Trihydrate and Cilnidipine in fixed-dose combination. The isocratic elution was accomplished by Symmetry C18 column (150 mm × 4.6 mm, 5 µm) at 25 °C. Mobile phase composition is Methanol: Acetonitrile: Potassium Dihydrogen Phosphate buffer (pH 3) (60:05:35%v/v/v) at a flow rate of 1.0 mL/min, injection volume 20 µL with DAD detection at 240 nm. Fimasartan Potassium Trihydrate and Cilnidipine were eluted with retention time 2.65 min and 5.51 min respectively. This method was validated as per ICH guideline (Q2 R1). The calibration plots were over the concentration range of 15–90 μg/mL and 2.5–15 μg/mL for Fimasartan Potassium Trihydrate and Cilnidipine with correlation coefficient 0.9992 and 0.9989 respectively. Accuracy was obtained between 99.51–101.65% and 100.06–101.20% for Fimasartan Potassium Trihydrate and Cilnidipine respectively. LOD were found to be 0.97 μg/mL and 0.57 μg/mL and LOQ were found to be 2.95 μg/mL and 1.75 μg/mL for Fimasartan Potassium Trihydrate and Cilnidipine respectively. The results showed that the developed method is reliable for the routine analysis for simultaneous determination of Fimasartan Potassium Trihydrate and Cilnidipine.


Background
Hypertension is defined as a sustained increase in blood pressure ≥ 140/90 mm Hg, a criterion where the risk of hypertension-related cardiovascular disease is high enough to merit medical attention [1]. It is the most common cardiovascular disease. In India, 29.8% population is suffering from hypertension. A systolic pressure above 140 or a diastolic pressure above 90. Blood pressure is the lateral pressure exerted by blood on the wall of the blood vessel while flowing through it. It's produced due to cardiac output and peripheral resistance [1].
Cilnidipine, a newer L/N-type Calcium channel blocker, is also an effective antihypertensive. It resolves the amlodipine induced pedel edema while maintaining adequate control of blood pressure [3]. It is chemically, 3-(2-methoxyethyl) 5-(2E)-3-phenylprop-2-en-1-yl 2, 6-dimethyl-4-(3-nitrophenyl)-1, 4-dihydropyridine-3, 5-dicarboxylate. The reported solubility found in Acetonitrile and methanol [4]. Cilnidipine is used along with proper diet and regular exercise to control high blood pressure. Cilnidipine presents a very rapid absorption with a maximum peaked concentration after 2 h. Its distribution tends to be higher in the liver as well as in kidneys, plasma and other tissues [5]. The structure of the Cilnidipine is given in Fig. 2.

Materials and instrumentation
Fimasartan Potassium Trihydrate was obtained from Ajanta Pharma Limited, Aurangabad. Cilnidipine was also provided by a reputed company. HPLC grades Methanol, Acetonitrile, and Milli Q water were used. All other chemicals were analytical reagent grade. Chromatographic analysis was carried out using an Agilent Technologies HPLC, DAD detector, and EZ Chrome software for data acquisition. A 0.45 μm Nylon filter was used for filtration.

Chromatographic condition
The Methanol, Acetonitrile, and Phosphate buffer pH 3.0 in the ratio of 60:05:35%v/v/v was used as a mobile phase. The flow rate was maintained at 1.0 mL/min. The injection volume is kept 20 μL and the detection was carried out at 240 nm.

Preparation of standard stock solution
A standard stock solution of 100 μg/mL for Fimasartan Potassium Trihydrate and Cilnidipine was prepared by using methanol as diluent.

Preparation of working solution of the mixture of Fimasartan Potassium Trihydrate and Cilnidipine
A working solution of 30 μg/mL for Fimasartan Potassium Trihydrate and 5 μg/mL for Cilnidipine were prepared by appropriate dilution.

Preparation of mobile phase
Prepare a mixture of Methanol, Acetonitrile and Potassium Dihydrogen Phosphate buffer in the volume ratio 60:05:35%v/v/v for method optimization. Mix well and sonicate to degas the mixture. Adjust pH 3.2 with Orthophosphoric acid.

Method validation
Analytical validation parameters for the analysis of the proposed method were determined according to ICH (Q2 R1) guideline.
[21] The parameters such as

System suitability
The typical values for evaluating system suitability of a chromatographic procedure include the RSD < 1%, Asymmetry factor < 2, and Theoretical plates > 2000. The determination of system suitability of the analytical method was accomplished by assaying three samples of the same strength as Fimasartan Potassium Trihydrate and Cilnidipine. The sample concentration of Fimasartan and Cilnidipine used in this analysis was 30 μg/mL and 5 μg/mL respectively. The retention time, peak area, theoretical plates, and asymmetry factor were evaluated for system suitability.

Linearity
The linearity response was determined by analysing 06 independent level of calibration curve in the range of 15-90 µg/mL (15,

Accuracy and precision
The accuracy of the method was performed in triplicate at three different concentration levels of 50, 100 and 150% of the targeted concentration of drugs. The accuracy of the method was evaluated by calculating percentage recovery. Repeatability was performed under 6 replicates at the assay concentration of FIK and CLN. Intra-day and inter-day variations of FIK and CLN were performed in triplicate at three different concentration levels of 50, 100, and 150%. Results are expressed in the form of RSD.

Specificity
Specificity was performed by injecting diluent, placebo and sample solution to check the interference of excipients.

LOD and LOQ
The limit of detection (LOD) and limit of quantification (LOQ) were calculated by formula as given in ICH (Q2R1).

Robustness
The robustness of the method was established by introducing small deliberate change in experimental conditions like flow rate, pH of Mobile phase and Mobile phase LOD = 3.3 × Standard deviation/Slope LOQ = 10 × Standard deviation/Slope composition. The changes made in flow rate ± 0.1 mL/ min, for pH of Mobile phase ± 0.2 and mobile phase composition ± 2%.

Assay
The combination of Fimasartan Potassium Trihydrate (60 mg) and Cilnidipine (10 mg) (Film Coated Tablet) is approved by CDSCO for clinical Trial Phase-III trial. So, there is no any single marketed pharmaceutical formulation is available.
The sample (synthetic mixture) was analyzed for assay containing 60 µg/mL of FIK and 10 µg/mL of CLN by optimized HPLC method. (conc. or dose ratio is suggested by CDSCO) [23].

Optimized condition for method development
Various mobile phase compositions, different columns, pH, flow rate were investigated and the best result was achieved on Symmetry C 18 (150 mm × 4.6 mm, 5 μm) column with the mobile phase composition was Methanol: Potassium Dihydrogen Phosphate buffer: Acetonitrile (60:35:05% v/v/v) pH 3 adjusted with OPA. The detection was carried out at 240 nm with 1.0 mL/min flow rate. The retention time of FIK and CLN is 2.65 and 5.51 min respectively with a total run time of 10 min. The optimized chromatogram and optimized conditions are mentioned in Fig. 3 and Table 1.

System suitability parameter
The optimized method is acceptable as system suitability parameters are valid as it passed the criteria of acceptability, as shown in Table 2.

Method validation Linearity
The calibration curve obtained for FIK and CLN in the range of 15-90 μg/mL and 2.5-15 μg/mL and the correlation coefficient was found to be 0.9992 and 0.9989 respectively. Linearity graph of both the drugs is revealed in Additional file 1: Figs. S1 and S2 and Additional file 2: Table S1.

Accuracy
This method is accurate as % Recovery was found in the range of 99.51-101.65% for FIK, while for CLN it was found to be in the range of 100.06-101.20% are shown in Tables 3 and 4.

Precision
Repeatability and intermediate precision express in term of RSD. As RSD was found to be < 2 indicating the presented method is precise. The results are summarized in

LOD and LOQ
LOD and LOQ were found to be 0.97 μg/mL and 2.95 μg/mL for FIK and 0.57 μg/mL and 1.75 μg/mL for CLN respectively.

Robustness
Making a deliberate change in flow rate, pH of mobile phase and mobile phase composition were taken place and RSD was found to be less than 2, specify that the method is robust. Results, presented in Tables 5, 6, and 7 indicate that the selected factors remained unaffected by a small variation of these parameters.

Assay of synthetic mixture
A synthetic mixture of FIK & CLN containing 60 mg of FIK and 10 mg of CLN when analysed using the developed method, showed 100.31% assay for FIK and 100.01% assay for CLN. The combination of Fimasartan Potassium Trihydrate and Cilnidipine (Film Coated Tablet) is approved by CDSCO for clinical Phase-III trial. So, there is no any single marketed pharmaceutical formulation is available. The results are tabulated in Table 8.

Discussion
The present method was developed using Methanol: Acetonitrile: Potassium Dihydrogen Phosphate buffer at pH 3.0 adjusted with diluted OPA in the ratio of 60:05:35% v/v/v. The method was developed with the minimum or reduced amount of organic solvents as the mobile phase which results in a more sensitive and cost-effective method. In the present method, Fimasartan Potassium Trihydrate and Cilnidipine were eluted with retention time 2.65 min and 5.51 min respectively. On the basis

Conclusion
A simple, rapid HPLC method was developed for simultaneous determination of Fimasartan Potassium Trihydrate and Cilnidipine in the synthetic mixture, which provides sufficient resolution between both drugs. The developed method was validated by testing its linearity, specificity, accuracy, precision, robustness and results were found within the acceptance limit as per ICH (Q2 R1) guideline. Statistical analysis proves that the method is suitable for the simultaneous determination of both drugs in pharmaceutical analysis and routine quality control without any interference.
Additional file 1: Figure S1. The calibration standard curve has been performed for Linearity determination of FIK (Pure Form) in the concentration range 15-90 μg/mL. Figure S2. The calibration standard curve has been performed for Linearity determination of CLN (Pure Form) in the concentration range 2.5-15 μg/mL. Figure S3. In the Specificity determination, the chromatogram of Blank is performed for the comparison with sample solution. Figure S4. The chromatogram of placebo is performed for the comparison with sample solution for determination of Excipients interference. Figure S5. Excipients interference is not observed at the working wavelength of 240 nm, as % Interference was found less than 0.6% for both drugs. It is proven by comparing chromatograms of blank, Placebo and sample preparation solution; there was no interference of excipients with the peak of FIK and CLN.
Additional file 2: Table S1. The calibration curve obtained for FIK in the range of 15-90 μg/mL and the correlation coefficient was found to be 0.9992 . Table S2. The calibration curve obtained for CLN in the range of 2.5-160 15 μg/mL and the correlation coefficient was found to be 0.9989. Table S3. Repeatability study and Intermediate precision has been performed according to guidelines and RSD was found to be <2 indicating the presented method is precise. Table S4. Specificity for FIK has been performed with and without spiking of Placebo; there was no interference of excipients with the peak of FIK. Table S5. Specificity for CLN has been performed with and without spiking of Placebo; there was no interference of excipients with the peak of CLN. Thus, the method is Specific.